Engineered Dll4-overexpressing osteocyte-derived exosomes enhanced bone regeneration by regulating osteogenesis and angiogenesis.

Abstract

Delayed fracture healing often results from impaired osteocyte network reconstruction and inadequate vascularization. Our prior work demonstrated that osteocytes engineered to overexpress Dll4 (Dll4-osteocytes) exert dual pro-osteogenic/angiogenic effects. Thus, this study explores the exosomes derived from Dll4-osteocytes (Dll4-Exo) as a cell-free strategy to coordinate bone-vascular regeneration and accelerate repair.Dll4-Exo were isolated from lentivirus-transduced Dll4-osteocytes. Mouse bone marrow stromal cells (ST2 cells) and human umbilical vein endothelial cells (HUVECs) were treated with Dll4-Exo to evaluate osteogenesis (ALP staining, mineralization, qRT-PCR) and angiogenesis (scratch/transwell migration, tube formation). Notch dependence was tested with γ-secretase inhibitor DAPT., Dll4-Exo was locally administered in a mouse tibial fracture model. Healing was assessed via X-ray imaging, histology, immunohistochemistry, and immunofluorescence staining at days 14, 21, and 28. Exosomal miRNA profiles were analyzed by sequencing, and miR-23a-5p function was validated through mimic/inhibitor transfections.Dll4-Exo activated Notch signaling in ST2 cells, significantly upregulating osteogenic genes (Alpl: 9.4-fold increase; mineralization: 62% increase) and enhancing HUVEC migration (2.6-fold) and tube formation. In the fracture model, Dll4-Exo accelerated callus formation, improved bone remodeling (OCN: 1.52-fold increase), and promoted revascularization (CD31⁺ vessel density: 1.56-fold increase with enhanced maturity). Through miRNA sequencing, miR-23a-5p was identified as the most enriched miRNA in Dll4-Exo, which was functionally transferred to both ST2 cells (3.0-fold increase) and HUVECs (2.7-fold increase). Mechanistic studies demonstrated that the pro-osteogenic effect of Dll4-Exo is exerted by miR-23a-5p via Notch signaling activation in ST2 cells, whereas its pro-angiogenic effect on HUVECs occurs through miR-23a-5p-independent mechanisms.Dll4-Exo carrying miR-23a-5p activates Notch-dependent osteogenesis in ST2 cells, while stimulating angiogenesis in HUVECs through alternative mechanisms, synergistically accelerating fracture healing and osteocyte network reconstruction. This engineered exosome platform represents a clinically viable strategy for bone regeneration.